US9353166B2ExpiredUtilityA1

Chimeric peptides for the regulation of GTPases

65
Assignee: CHILDRENS MEDICAL CENTERPriority: Aug 13, 2003Filed: Mar 17, 2014Granted: May 31, 2016
Est. expiryAug 13, 2023(expired)· nominal 20-yr term from priority
Inventors:Yi ZhengDavid A. Williams
A61P 43/00A61P 35/00C12N 9/14C07K 14/4706C12Y 306/05002C07K 2319/01C07K 2319/02C07K 2319/03
65
PatentIndex Score
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Cited by
88
References
35
Claims

Abstract

Chimeric peptides or fusion proteins are disclosed that include a RhoGAP activity domain and at least one specificity domain that targets a specific Rho protein. The fusion proteins can be used to inhibit any GTPase activity within a cell. The fusion proteins are particularly advantageous for the treatment of cancer. The present invention generally relates to chimeric peptides capable of regulating GTPases, and more particularly, to methods of targeting individual GTPases by using GTPase-activating proteins. Such proteins may be used for the treatment of cancers and other GTPase-related diseases. This invention relates to nucleic acid molecules and the encoded GTPase activating proteins, and variants thereof, and to the use of these molecules in the characterization, diagnosis, prevention, and treatment of cell signaling, immune, and cell proliferative disorders, particularly cancer. Disclosed herein are compounds and methods for regulating transcription of a selected gene.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A method of treating a disease related to up-regulation of GTPase activity in a mammal, comprising administering to the mammal a composition in an amount effective to reduce the GTPase activity, wherein the composition comprises: (i) a chimeric polypeptide comprising a guanosine-triphosphatase-activating protein (GAP) activity domain consisting of amino acids 1248 to 1513 of SEQ ID NO: 2 or a fragment thereof having GAP activity, and at least one targeting domain comprising the amino acid sequence of SEQ ID NO: 16 or a fragment thereof which targets at least one specific guanosine triphosphatase (GTPase) protein; and (ii) a pharmaceutically acceptable carrier. 
     
     
       2. The method of  claim 1 , wherein the fragment of the GAP-activity domain having GAP activity consists of amino acids 1261 to 1431 of SEQ ID NO: 2. 
     
     
       3. The method of  claim 1 , wherein the chimeric polypeptide comprises an amino acid sequence of SEQ ID NO: 9. 
     
     
       4. The method of  claim 3 , wherein the chimeric polypeptide consists of an amino acid sequence of SEQ ID NO: 9. 
     
     
       5. The method of  claim 1 , wherein the targeting domain is a C-terminal sequence of a GTPase. 
     
     
       6. The method of  claim 1 , wherein the targeting domain is N-terminal to the GAP activity domain. 
     
     
       7. The method of  claim 1 , wherein the targeting domain is C-terminal to the GAP activity domain. 
     
     
       8. The method of  claim 1 , wherein the chimeric polypeptide further comprises a membrane-translocating protein domain or a membrane-translocating peptide. 
     
     
       9. The method of  claim 8 , wherein:
 (i) when the chimeric polypeptide comprises a membrane-translocating peptide, the membrane-translocating peptide comprises a membrane-associated isoprenylation modification; or 
 (ii) when the chimeric polypeptide comprises a membrane-translocating protein domain, the membrane-translocating protein domain comprises a lipid-modified peptide. 
 
     
     
       10. The method of  claim 8 , wherein the chimeric polypeptide comprises a membrane-translocating peptide, and the membrane-translocating peptide is an amino acid sequence having about 8 amino acids to about 24 amino acids comprising at least 8 consecutive amino acids of an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. 
     
     
       11. The method of  claim 8 , wherein chimeric polypeptide comprises a membrane-translocating protein domain, wherein the membrane-translocating protein domain is a peptide of about 8 amino acids to about 50 amino acids. 
     
     
       12. A method for modulating GTPase activity in a cell, comprising: contacting the cell with a chimeric polypeptide that modulates GTPase activity, wherein the chimeric polypeptide comprises a guanosine-triphosphatase-activating protein (GAP) activity domain consisting of amino acids 1248 to 1513 of SEQ ID NO: 2 or a fragment thereof having GAP activity, and at least one targeting domain comprising the amino acid sequence of SEQ ID NO: 16 or a fragment thereof which targets at least one specific guanosine triphosphatase (GTPase) protein. 
     
     
       13. The method of  claim 12 , wherein the fragment of the GAP-activity domain having GAP activity consists of amino acids 1261 to 1431 of SEQ ID NO: 2. 
     
     
       14. The method of  claim 12 , wherein the chimeric polypeptide comprises of an amino acid sequence of SEQ ID NO: 9. 
     
     
       15. The method of  claim 14 , wherein the chimeric polypeptide consists of an amino acid sequence of SEQ ID NO: 9. 
     
     
       16. The method of  claim 12 , wherein the targeting domain is a C-terminal sequence of a GTPase. 
     
     
       17. The method of  claim 12 , wherein the targeting domain is N-terminal to the GAP activity domain. 
     
     
       18. The method of  claim 12 , wherein the targeting domain is C-terminal to the GAP activity domain. 
     
     
       19. The method of  claim 12 , wherein the chimeric polypeptide further comprises a membrane-translocating protein domain or a membrane-translocating peptide. 
     
     
       20. The method of  claim 19 , wherein:
 (i) when the chimeric polypeptide comprises a membrane-translocating peptide, the membrane-translocating peptide comprises a membrane-associated isoprenylation modification; or 
 (ii) when the chimeric polypeptide comprises a membrane-translocating protein domain, the membrane-translocating protein domain comprises a lipid-modified peptide. 
 
     
     
       21. The method of  claim 19 , wherein the chimeric polypeptide comprises a membrane-translocating peptide, and the membrane-translocating peptide is an amino acid sequence having about 8 amino acids to about 24 amino acids comprising at least 8 consecutive amino acids of an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. 
     
     
       22. The method of  claim 19 , wherein chimeric polypeptide comprises a membrane-translocating protein domain, wherein the membrane-translocating protein domain is a peptide of about 8 amino acids to about 50 amino acids. 
     
     
       23. A method for the treatment of cancer in a patient, comprising administering a composition to the patient in an amount effective to inhibit GTPase activity, wherein the composition comprises: (i) a chimeric polypeptide comprising a guanosine-triphosphatase-activating protein (GAP) activity domain consisting of amino acids 1248 to 1513 of SEQ ID NO: 2 or a fragment thereof having GAP activity, and at least one targeting domain comprising the amino acid sequence of SEQ ID NO: 16 or a fragment thereof which targets at least one specific guanosine triphosphatase (GTPase) protein; and (ii) a pharmaceutically acceptable carrier. 
     
     
       24. The method of  claim 23 , wherein the cancer is characterized by increased activity of a GTPase. 
     
     
       25. The method of  claim 23 , wherein the cancer is selected from the group consisting of: squamous cell cancer, head and neck cancer, and breast cancer. 
     
     
       26. The method of  claim 23 , wherein the fragment of the GAP-activity domain having GAP activity consists of amino acids 1261 to 1431 of SEQ ID NO: 2. 
     
     
       27. The method of  claim 23 , wherein the chimeric polypeptide comprises an amino acid sequence of SEQ ID NO: 9. 
     
     
       28. The method of  claim 27 , wherein the chimeric polypeptide consists of an amino acid sequence of SEQ ID NO: 9. 
     
     
       29. The method of  claim 23 , wherein the targeting domain is a C-terminal sequence of a GTPase. 
     
     
       30. The method of  claim 23 , wherein the targeting domain is N-terminal to the GAP activity domain. 
     
     
       31. The method of  claim 23 , wherein the targeting domain is C-terminal to the GAP activity domain. 
     
     
       32. The method of  claim 23 , wherein the chimeric polypeptide further comprises a membrane-translocating protein domain or a membrane-translocating peptide. 
     
     
       33. The method of  claim 32 , wherein:
 (i) when the chimeric polypeptide comprises a membrane-translocating peptide, the membrane-translocating peptide comprises a membrane-associated isoprenylation modification; or 
 (ii) when the chimeric polypeptide comprises a membrane-translocating protein domain, the membrane-translocating protein domain comprises a lipid-modified peptide. 
 
     
     
       34. The method of  claim 32 , wherein the chimeric polypeptide comprises a membrane-translocating peptide, and the membrane-translocating peptide is an amino acid sequence having about 8 amino acids to about 24 amino acids comprising at least 8 consecutive amino acids of an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. 
     
     
       35. The method of  claim 32 , wherein chimeric polypeptide comprises a membrane-translocating protein domain, wherein the membrane-translocating protein domain is a peptide of about 8 amino acids to about 50 amino acids.

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